The objective of this core TR&D project is to improve methods for the selective staining of neurons for correlative light and electron microscopy and to develop methods that will help researchers to visualize and quantify neuronal shape and connectivity at EM resolution. Our efforts in this area have been driven by our collaborative efforts with Drs. Charles Wilson, Gordon Arbuthnott and Cali Ingham on obtaining quantitative information on structural changes in striatal dendrites and dendritic spines associated with partial deafferentation. Briefly, we have been using electron microscopic tomography to obtain volume reconstructions of spiny dendrites in both normal neostriatum and in neostriatum in which the doparninergic input has been lesioned chemically. Spiny dendrites receive the bulk of dopaminergic input within the neostriatum and it was observed at the light microscopic level that loss of this input may lead to both a loss of spines and a change in their morphology. Considerable effort has been expended upon this project with resulting improvements in specimen preparation, tomographic reconstruction techniques and computer-aided analysis. This year, we were focused on collecting and analyzing data from lesioned and unlesioned neostriatal dendrites. Tomographic reconstructions have been obtained from 20 dendrites that were selected from the same region of Golgi-impregnated neurons from the lesioned and unlesioned neostriata of 4 animals. The dendrite was segmented from the grayscale volume using Xvoxtrace, a tracing program developed at the NCMIR that allows the contours traced on an individual volume slice to be displayed on the 3-dimensional volume and the tilt series used to construct the volume. Copies of Xvoxtrace have been supplied to our collaborators, Dr. Cali Ingham and Dr. Charlie Wilson, so that they can participate in the tracing at their home institutions. Once the dendrltes have been traced, they are converted into a format readable by Analyze, a program for displaying and measuring 3-dimensional volumes. Measures of the surface area and volume of individual spines are obtained and recorded. Thus far, we have completed measurements of 14 out of the 20 dendrites. This analysis is being performed blindly so we don't know which dendrites come from the lesioned or unlesioned striata. Once all of the data is obtained, we will break the code and determine whether any differences in spine parameters exists between the lesioned and unlesioned sides. We have also included in the 20 reconstructions, 3 dendrites from different neurons in the same striatum to get a measure of inter-neuronal variability. We also have obtained 2 dendrites from each of two neurons to obtain a measure of intradendritic variability in a single neuron. Once this initial analysis is complete, we will determine whether additional datasets are necessary for this study. We have also been using tomographic reconstruction techniques to obtain surface area and volume measurements of more complicated structures such as astrocytic processes. Several such reconstructions have been performed in collaboration with Dr. Kyoshi Hama. We anticipate that during the next year that we will begin to perform tomographic reconstructions on neurons that have been intracellularly injected with a fluorescent dye and photoconverted for correlated light and electron microscopic analysis as parts of several projects designed to provide accurate morphological compartrnental models of actual neurons for neuromcdeling studies. We are still pursuing additional staining techniques for cells and their processes. We have been using immunocytochemical and~Golgi staining to delineate the basket cell axonal ramification aroung the Purkinje cell initial segment, the pinceau. We are currently performing three dimensional reconstructions of this interesting structure usign serial section and tomographic reconstruction to quantify the amount of contact between the basket cell fibers and the initial segment. Another project involves using the enzyme reaction for i' nucleosidase to study the patch-matrix organization in the rat neostriatum. This enzyme fills the extracellular space and should prove an interesting stain for the use of tomography to investigate the structure of the neuropil.